Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-P&F.19] Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system Borys Chong-Pérez 1,2,∗ , Geert Angenon 2 , Rafael G Kosky 1 , Maritza Reyes 1 , Luis E. Rojas 1 1

mined by PCR (Figure 1B) and Southern hybridization, was 59.7 and 44.0% for GmHSP17.6-L and AtHSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. The system described here is simple and might be extensively applicable for the production of markerfree transgenic plants of many crop species.

Instituto de Biotecnología de las Plantas, Universidad Central “Marta Abreu” de Las Villas. Carretera a Camajuaní km 5.5, Santa Clara, Cuba 2 Laboratory of Plant Genetics, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel, B–1050 Brussels, Belgium Keywords: Agrobacterium tumefaciens; auto-excision; genetic transformation; Musa

doi:10.1016/j.jbiotec.2010.09.723

Selectable marker genes are frequently used for transgenic event production, but after the selection process these genes are not longer needed. Moreover, they may cause public concern and technological problems. Several excision systems exist but few have been optimized or shown to be functional for clonally propagated crops. Marker free transgenic banana plants cv. Grande Naine (Musa AAA) have been obtained using a Cre/loxP auto-excision strategy. We used a binary expression vector in which the recombinase gene cre under the control of a heat shock promoter and selectable markers genes cassettes were placed between two loxP sites in direct orientation, while the gene of interest was inserted outside of the loxP sites (Figure 1A). Heat shock promoters GmHSP17.6-L

Piyachat Wiriyaampaiwong 1,∗ , Pornthap Thanonkeo 1 , Sudarat Thanonkeo 2

[P-P&F.20] Cloning and Characterization of Chalcone Synthase Gene from Pueraria candollei var. mirifica

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Khon Kaen University, Thailand Mahasarakham University, Thailand Keywords: Pueraria candollei var. mirifica; Chalcone synthase; Flavonoid biosynthesis 2

Pueraria candollei var. mirifica is an indigenous herb found in the North, West and Northeastern part of Thailand. This plant synthesized and accumulated several biological active compounds namely phytoestrogen such as miroestrol, deoxymiroestrol, daidzin, puerarin, genistin, mirificin, kawakhurin, coumestrol, daidzein and genistein. Chalcone synthase (CHS; EC 2.3.1.74), which catalyzes the formation of chalcone from p-coumaroyl and malonyl CoAs, is the key enzyme in flavonoid biosynthesis in plant. In this study, cloning and characterization of chalcone synthase gene (CHS) from P. candollei were carried out. Using a reverse transcriptionpolymerase chain reaction (RT-PCR) strategy, the CHS gene was cloned from P. candollei. The complete PcCHS coding sequence contained 1170 bp, encoded for a polypeptide of 389 amino acid residues with a calculated molecular mass of 42.8 kDa and pI of 5.31. Southern blot hybridization of genomic DNA fragments revealed the presence of multiple CHS genes in the P. candollei genome. Homology analysis revealed that the deduced amino acid sequence of PcCHS had 86-93% identity, whereas the nucleotide sequence had 81-95% identity to CHS of other plants, such as P. montana, Glycine max, Phaseolus vulgaris, Vigna unguiculata, Pisum sativum and Medicago sativa. Expression analysis of the PcCHS gene in different plant tissues by RT-PCR revealed that this gene is expressed in a tissuespecific manner. The expression of the PcCHS gene was remarkably induced by high temperature, UV-B and wounding treatments, suggesting that the CHS gene product may plays a crucial role in protecting plant from environmental or external stresses. The results in this study provide useful information for further work on production of phytoestrogen, an active compound in this plant. doi:10.1016/j.jbiotec.2010.09.724 [P-P&F.21]

Fig. 1. Outline of the marker removal strategy and PCR analysis. A, Schematic representation of the original T-DNA used for transformation (HCN) and the excision fragment after Cre-mediated recombination (N). B, PCR results. PCR1, primers C-3300-F/nptII-SR yield a 724 bp fragment specific for the excision allele; PCR2, primers codA-1/nptII-SR yield a 1441 bp fragment specific for the original allele. Lanes 1 to 16, DNA purified from transgenic plants submitted to heat shock; C-, untransformed plant; pGmhsp-A, plasmid control; H2 O, water. M: Molecular weight marker Gene RulerTM DNA Ladder Mix (Fermentas).

and AtHSP18.2, from soybean and Arabidopsis respectively, were tested. The results showed that a transient heat shock treatment of primary transgenic embryos is sufficient for inducing cre and excising the cre, hpt and codA genes. Excision efficiency, as deter-

Assessing genetic diversity of Tamarix spp. in three populations in Southern Italy S. Terzoli ∗ , G. Abbruzzese, I. Beritognolo, M. Sabatti, R. Valentini, E. Kuzminsky University of Tuscia, Italy Keywords: Tamarix; Microsatellite; Genetic variability; Abiotic stress Tamarix plants are resistant to abiotic stresses, as they thrive in zones where drought, soil salinity, and high temperature are